The long-term objective of this application is to develop methods for the efficient determination of atomic-resolution three-dimensional (3D) structures of large biological complexes by cryo electron microscopy (cryoEM). In the current funding period, the PI's group has developed data acquisition and processing methods, implemented software, and validated these advances by determination of cryoEM structures of a number of large icosahedral and helical complexes, as well as complexes of low or no symmetry, some to an unprecedented 2.9A resolution. We hypothesize that further advances in cryoEM and single-particle reconstruction can be obtained by taking advantage of electron counting and super-resolution capabilities of direct electron detectors for determination of atomic models for even larger or transiently stable complexes. Whereas we aimed and succeeded in the current funding period to obtain cryoEM structures at atomic resolution for icosahedral and helical viruses, the goal of this renewal application is to develop methods for atomic structures for even larger complexes, including those that are only transiently stable. In the renewal, we propose to improve both imaging itself and computation to bring resolution to ~2A, permitting identification of small ligands or drugs in large complexes, even resolving holes of benzene rings of aromatic side chains. Specifically, we will develop novel imaging and image correction methods that take advantage of the practically noise-free images afforded by a newly installed direct electron detector (Aim #1); achieve high-resolution reconstructions of complexes that are deformable or intrinsically structurally heterogeneous by a symmetry- based selection method (Aim #2); test use of de novo atomic models to refine orientation and center parameters to further improve map resolution and model accuracy (Aim #3); and validate these new methods for atomic structure determination by application to the icosahedral complexes cytoplasmic polyhedrosis virus (CPV), herpesvirus capsids and membrane-containing dengue virus, filamentous complexes including tobacco mosaic virus (TMV), NS1 tubule and actin, and other biological machines such as pyocin, pyruvate dehydrogenase and telomerase complexes (Aim #4). A successful outcome of this renewal project will further push the resolution limit of cryoEM in structural studies of large complexes and will have great impact on many areas of biomedical research. This achievement will complement other structural methods, particularly X-ray crystallography of purified proteins that are amenable to crystallization and NMR of small molecules in solution. Specifically, this achievement will enable investigators to place individual proteins within the structural context of the larger assembly and to visualize native shapes and physical chemical interactions among the parts of the complex. Moreover, the ability to look at large complexes at atomic resolution will permit visualization of complexes bound to antibodies.